Christophe Nguyen-The

The microbiology of minimally processed fresh fruits and vegetables

Minimally processed fresh (MPF) fruits and vegetables are good media for growth of microorganisms. They have been involved in outbreaks because of the consumption of products contaminated by pathogens. They are also sensitive to various spoilage microorganisms such as pectinolytic bacteria, saprophytic Gram-negative bacteria, lactic acid bacteria, and yeasts. Contamination of MPF fruits and vegetables occurs at every stage of the food chain, from cultivation to processing. Polluted environments during cultivation or poor hygienic conditions in processing increase the risk of contamination with foodborne pathogens. Although MPF fruits and vegetables may harbor psychrotrophic microorganisms such as fluorescent pseudomonads or Listeria monocytogenes, good control of refrigeration temperature limits growth of spoilage and pathogenic microorganisms. Modified atmospheres are often efficient to maintain or improve visual organoleptic quality of MPF fruits and vegetables, but their effects on microorganisms are inconsistent. Chemical disinfection can partially reduce the initial bacterial contamination; irradiation seems to be more efficient. The applications of legislations and quality assurance systems to control contamination, survival, and growth of foodborne pathogens in MPF fruits and vegetables are discussed.

Enterotoxigenic Profiles of Food-Poisoning and Food-Borne Bacillus cereus Strains

ABSTRACT The enterotoxigenic profiles of 51 B. cereus food-related strains were compared to those of 37 B. cereus food-poisoning strains. cytK and association of hbl-nhe-cytK enterotoxin genes were more frequent among diarrheal strains (73 and 63%) than among food-borne strains (37 and 33%). Unlike diarrheal strains, food-borne strains showed frequent nhe and hbl gene polymorphisms and were often low toxin producers.

Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus Group occasionally associated with food poisoning.

An aerobic endospore-forming bacillus (NVH 391-98(T)) was isolated during a severe food poisoning outbreak in France in 1998, and four other similar strains have since been isolated, also mostly from food poisoning cases. Based on 16S rRNA gene sequence similarity, these strains were shown to belong to the Bacillus cereus Group (over 97% similarity with the current Group species) and phylogenetic distance from other validly described species of the genus Bacillus was less than 95%. Based on 16S rRNA gene sequence similarity and MLST data, these novel strains were shown to form a robust and well-separated cluster in the B. cereus Group, and constituted the most distant cluster from species of this Group. Major fatty acids (iso-C(15:0), C(16:0), iso-C(17:0), anteiso-C(15 : 0), iso-C(16:0), iso-C(13:0)) supported the affiliation of these strains to the genus Bacillus, and more specifically to the B. cereus Group. NVH 391-98(T) taxon was more specifically characterized by an abundance of iso-C(15:0) and low amounts of iso-C(13:0) compared with other members of the B. cereus Group. Genome similarity together with DNA-DNA hybridization values and physiological and biochemical tests made it possible to genotypically and phenotypically differentiate NVH 391-98(T) taxon from the six current B. cereus Group species. NVH 391-98(T) therefore represents a novel species, for which the name Bacillus cytotoxicus sp. nov. is proposed, with the type strain NVH 391-98(T) (= DSM 22905(T) = CIP 110041(T)).

Prevalence, characterization and growth of Bacillus cereus in commercial cooked chilled foods containing vegetables.

In cooked‐chilled and pasteurized vegetable products, initial numbers of Bacillus cereus were below 10 cfu g−1. Before the appearance of spoilage, numbers reached 6–8 log cfu g−1 at 20 °C and 4–6 log cfu g−1 at 10 °C. Bacillus cereus was not detected in samples stored at 4 °C. Ten percent of strains isolated from the products were able to grow at 5 °C and 63% at 10 °C. Bacillus cereus strains unable to degrade starch, a feature linked to the production of emetic toxin, did not grow at 10 °C and had a higher heat resistance at 90 °C. Using immunochemical assays, enterotoxin was detected in the culture supernatant fluid of 97·5% of the strains. All culture supernatant fluids were cytotoxic but important variations in the level of activity were found. Psychrotrophic isolates of B. cereus were unable to grow in courgette broth at 7 °C whereas they grew in a rich laboratory medium. At 10 °C, these isolates grew in both media but lag time in courgette broth was 20‐fold longer than in the rich laboratory medium.

Influence of Background Microflora on Listeria monocytogenes on Minimally Processed Fresh Broad-Leaved Endive (Cichorium endivia var. latifolia)

Interactions between Listeria monocytogenes and the background microflora of endive leaves was studied by (i) testing the effect of disinfecting endive leaves on the fate of L. monocytogenes , and (ii) challenging L. monocytogenes along with each of 10 strains of pseudomonads and Enterobacteriaceae isolated from endive leaves on both leaves of endive and in a model sterile medium based on an exudate of endive leaf. There was a higher increase in the population of L. monocytogenes on endive leaves with a background microflora reduced by a chemical disinfection. High numbers (106 to 107 CFU/g) of some strains reduced the growth of L. monocytogenes on endive. None of the 10 strains tested promoted the growth of L. monocytogenes . In the medium made of endive leaf exudate, the 10 strains tested reduced the maximum growth of L. monocytogenes to extents that varied with the strains. A complex bacterial population extracted from endive leaves completely inhibited the growth of L. monocytogenes in the medium.

Detection of depolymerase isoenzymes after electrophoresis or electrofocusing, or in titration curves☆

The cup-plate technique makes it possible to detect enzyme activities after diffusion into buffered, substrate-containing agar gels. This technique has been used after nondenaturing blotting transfer in order to detect depolymerizing enzyme activities once analytical protein separation (e.g., by electrophoresis, electrofocusing, or titration curves) has been completed. This rapid and very sensitive method was successfully applied to the enzymes polygalacturonate lyase, polygalacturonate hydrolase, endoglucanase, and xylan hydrolase. Other possible applications are presented.

Ability of Bacillus cereus group strains to cause food poisoning varies according to phylogenetic affiliation (groups I to VII) rather than species affiliation.

ABSTRACT Cytotoxic activity levels of culture filtrates and toxin distributions varied according to the phylogenetic group (I to VII) within the B acillus cereus group, suggesting that these groups are of different clinical significance and are more suitable than species affiliations for determining food poisoning risk. A first-line, simple online tool (https://www.tools.symprevius.org/Bcereus/english.php ) to assign strains to the different phylogenetic groups is presented.

Survival of Bacillus cereus spores and vegetative cells in acid media simulating human stomach

Aims:  To determine the fate of Bacillus cereus spores or vegetative cells in simulated gastric medium.

Effects of carbon dioxide on the fate of Listeria monocytogenes, of aerobic bacteria and on the development of spoilage in minimally processed fresh endive

Minimally processed fresh broad-leaved endive (Cichorium endivia L.) were stored at 3 and 10 degrees C in modified atmospheres containing air, 10% CO2/10% O2, 30% CO2/10% O2, and 50% CO2/10% O2. The effects of these modified atmospheres on the fate of both aerobic bacteria and three strains of Listeria monocytogenes, was investigated. Increases in CO2 concentrations significantly reduced the growth of the aerobic microflora. The best preservation of the visual quality occurred on endive leaves stored in 10% CO2/10% O2, whereas leaves stored in 30% CO2/10% O2 and 50% CO2/10% O2, and to a lesser extent in air, showed extensive spoilage after storage. Listeria monocytogenes was slightly affected at 3 degrees C by the modified atmospheres, as compared to air. At 10 degrees C, results varied between replicate experiments, but L. monocytogenes generally grew better as the CO2 concentration was increased. The three test strains behaved in a similar way. In conclusion, among the modified atmospheres tested, a modified atmosphere containing 10% CO2/10% O2 resulted in improved visual quality of minimally processed fresh endive, without a marked effect on the growth of the aerobic microflora or of L. monocytogenes.

Identification of Bacteria in Pasteurized Zucchini Purées Stored at Different Temperatures and Comparison with Those Found in Other Pasteurized Vegetable Purées

ABSTRACT One hundred nineteen isolates from a commercial zucchini purée stored at 4, 10, and 20 to 25°C were fingerprinted using repetitive sequence-based PCR (REP-PCR) and classified into 35 REP types. One representative isolate of each REP type was subsequently identified by API50CHB/20E profile and partial rrs gene sequence analysis. Nine REP types were misidentified by the API system. Strains were misidentified as being in the Bacillus circulans (group 2) API taxon or in taxa with a low number of positive API characters such as Brevibacillus brevis. A phylogenetic analysis pointed to one new species ofBacillus and three new species ofPaenibacillus among the misidentified REP types. Bacterial components in zucchini purée were compared phenotypically with those obtained in previous work on broccoli, carrot, leek, potato, and split pea purées, based on simple matching coefficient and unweighted pair group method with averages cluster analysis. Out of 254 strains, 69 strains previously identified as B. circulans (group 2) or B. circulans/B. macerans/B.polymyxa were assigned to a newPaenibacillus taxon phylogenetically related toP. azotofixans. Storage conditions at 4°C favored the development of “B. macroides/B. maroccanus” and Paenibacillus spp. in zucchini purées andPaenibacillus spp. in other purées. Storage conditions at 20 to 25°C favored the development of B. subtilis group (B. licheniformis and B. subtilis) and B. cereus group strains. At 10°C, Paenibacillus spp. were always present at high frequencies, whereas the occurrence of B. macroides/B. maroccanus (in zucchini purées), B. cereus, and B. pumilus varied with the experiment.